A long-term goal of our research is to elucidate the molecular mechanisms that control axon guidance in the developing vertebrate central nervous system (CNS). Floor plate-derived chemoattractants (e.g., Netrins) guide commissural growth cones/axons along a circumferential/transverse path from the dorsolateral spinal cord toward the ventral midline. In complementary fashion, chemorepellents (e.g., BMPs) secreted by the roof plate, situated at the dorsal midline, are likely to initially orient these axons away from dorsal regions of the spinal cord. Once commissural axons reach the ventral midline, contact-dependent interactions between commissural growth cones and floor plate cells facilitate midline crossing. Upon exiting the ventral midline, most commissural axons execute an orthogonal turn and, at least initially, project in the rostral direction alongside the floor plate. Consistent with the finding that commissural axons gain responsiveness to floor plate-associated chemorepellents (e.g., Slits and Semas) after they cross through the floor plate in vitro, these axons do not recross the midline. We recently used DiI labeling in both rodent and chick embryos to demonstrate that many commissural axons exhibit a complex pattern of growth on the contralateral side of the floor plate; decussated commissural axons travel along a diagonal path away from the ventral midline and into intermediate/dorsal regions of the spinal cord, and then execute a second rostral turn along a conserved boundary of ephrinB expression. Consistent with the ability of B-class ephrin ligands to mediate short-range repulsive interactions, including the collapse of commissural growth cones in vitro, perturbation of EphB (receptor)-ephrinB interactions in cultured mouse spinal cord preparations results in the inappropriate projection of commissural axons into more dorsal regions of the spinal cord. These findings suggest that opposed gradients of long range floor plate- and roof plate-derived chemorepellents operate in concert with the short-range effects of B-class ephrins to shape the complex contralateral commissural pathway. To test this hypothesis, commissural axon pathfinding on the contralateral side of the midline will be examined in floor plate- and roof plate-lacking mice, as well as in mice lacking Slit, Robo, NPN2, BMP7 or Eph receptors and ephrinB ligands. Furthermore, both in vitro and in ovo systems will be used to determine whether blocking Slit-Robo, Sema-NPN, Eph-ephrinB interactions and BMP/activity, or ectopically expressing ephrinB proteins, perturbs commissural axon pathfinding on the contralateral side of the spinal cord.